Millions of Americans suffer from dry eye, compromising ocular surface health and comfort. While dry eye has many causes, a significant number of cases are linked to decreased production of fluid and protein by the lacrimal gland (LG). Some of the most severe dry eye cases are associated with Sjgren's syndrome (SS), an autoimmune disease with lymphocytic infiltration of LG and salivary glands, alterations in volume and composition of exocrine secretions, and increased risk of B-cell lymphoma. While SS-associated dry eye affects 0.4-0.8% of the population, the mechanisms responsible for its initiation and progression are poorly understood. Therapies for SS-associated dry eye that move beyond management of symptoms to address the underlying LG inflammation and pathological changes in tear composition are simply not available. LG acinar cells (LGAC) produce and secrete most tear proteins, while the sorting of newly-synthesized tear proteins into secretory vesicles is governed by many effectors including Rabs, small membrane-associated GTPases. LGAC secretory vesicles are enriched in Rab3D, Rab27a and Rab27b, with their abundance varying across individual vesicles. Changes in Rab abundance on secretory vesicles may reflect differences in content, due to differing contributions of these Rabs in mediating traffic from distinct compartments of origin. Using knockout mouse strains, we have linked Rab27a and Rab27b to secretion of tear cathepsin S (CTSS). A potent lysosomal protease, CTSS has roles in antigen presentation, extracellular matrix remodeling and inflammation; we have found that CTSS is significantly elevated in tears and LG of a mouse model of SS and in tears of SS patients. We HYPOTHESIZE that 1) Tear CTSS is highly regulated through its trafficking via a novel Rab27-mediated pathway from lysosomes to SV; and 2) Exposure of the LG to inflammatory cytokines may elevate tear CTSS by increasing its expression in LG and/or by altering the expression/activity of secretory Rabs. Finally, increased tear and LG CTSS activity may promote the ocular surface inflammation and autoimmune dacryoadenitis that characterize SS-associated dry eye. Three AIMS are proposed: 1) Characterize the pathways responsible for sorting of CTSS into secretory vesicles in LGAC; 2) Determine how pro-inflammatory cytokines affect CTSS sorting and release from secretory vesicles in LGAC; and 3) Determine whether increased tear and LG CTSS drive ocular surface inflammation and autoimmune dacryoadenitis. These studies will utilize cultured LGAC for in vitro analysis of Rab function, including LGAC cultured from Rab knockout mice, using confocal fluorescence and super-resolution microscopy, analysis of protein secretion, and molecular analysis of gene expression, with and without exposure to pro-inflammatory cytokines. They will also utilize the male NOD mouse model of SS-associated dry eye to assess the ability of CTSS inhibitor to reduce signs of disease. Completion of these AIMS will 1) Define mechanisms underlying increased tear CTSS activity in SS patients; and 2) Determine if CTSS actively drives SS-associated dry eye.